DOCSLIB.ORG

Isolation and Characterization of an Alkaloid-Blocked Mutant of Claviceps Paspali

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Isolation and Characterization of an Alkaloid-Blocked Mutant of Claviceps Paspali J. Basic Microbiol. 30 (1990) 1,51-56 (“Boris KidriE” Institute of Chemistry, Ljubljana, Yugoslavia) Isolation and characterization of an alkaloid-blocked mutant of Claviceps paspali ELIZABETAPERTOT, VLADIMIRA GABERC-POREKAR and HELENASOCIC (Received 24 April I989/Accepted 3 May 1989) After UV-irradiation an alkaloid-blocked mutant of Claviceps pusgufi was isolated from an ergometrine and lysergic acid amides (1 : 1) producing strain. The formation of ergometrine is blocked in this particular mutant strain which accumulates only lysergic acid amides. A significant morphological differentation of colonies of mutant as well as of mycelium in submerged cultures could be observed and total alkaloid yields are rather influenced. The fungus Claviceps may be divided into numerous chemical races with different alkaloid spectra. Claviceps paspali strains in submerged cultures can produce : a-hydroxyethyl- amide of lysergic acid (ARCAMONEet al. 1961), ergometrine (GR~ERand TYLER1963) and A8*9-lysergicacid (KOBELet al. 1964), whereas several strains of C. purpurea produce mainly peptide alkaloids (GR~ERand ERGE 1970, SPALLA 1980, UDVARDY 1980, BIANCHIet a/. 1982). From the results of several investigators (KLEINEROVAand KYBAL 1969, KOBELet al. 1962, VOIGTand KEIPERT1967) it can be concluded that the composition of the alkaloid complex is genetically determined and that in nature biosynthetic pathway can proceed up to the peptide alkaloids, or be blocked more or less completely at some intermediate point. The spectra of saprophytically synthesized ergot alkaloids may be changed either by mutation (STRNADOVA1964, 1967, KOBELand SANGLIER1973, GR~GER1979) or by changing the culture conditions (KRENet al. 1987, BREUELet af. 1982). The literature concerning the mutational biosynthesis in Claviceps is very sparse. An alkaloid- blocked mutant was isolated from an ergotoxine-producing C. purpurea strain, which accumulates chanoclavine-I and the corresponding chanoclavine-I aldehyde (MAIERet al. 1980). Another interesting mutant, obtained by multiple mutagenesis with UV light, was described by KRENet al. (1986). The block in the biosynthetic pathway of this mutant was only partial, since beside chanoclavine-I and chanoclavine-I aldehyde also small amounts of tetracyclic clavines elymoclavine and agroclavine were produced. From a C. paspali lysergic acid a-hydroxyethylcarbinolamide producing strain a mutant capable of accumulating ergometrine was attained (MARNATIet al. 1975). In the present communication we describe another alkaloid-blocked mutant of C. paspali obtained after UV-irradiation. The mutant accumulates lysergic acid amides, while the formation of ergometrine is blocked. Materials and methods Microorganism : A Claviceps paspali (STEVENSand HALL)selected strain L-52, capable of producing simple lysergic acid derivatives, and from it derived the stable UV mutant strain designated as CP2, were employed in this study. Mutagenic treatment: A four-day old vegetative mycelium of C. paspali was homogenized in a WARiNG blendor for LO sec and mycelial fragments in 0.9% NaCl solution were exposed to UV irradiation (300 Jimz) 4’ 52 ELISABETAPERTOT et al, for time sufficient to kill 90-99% of the cells. Treated mycelial fragments were plate - cultured on potato- glucose agar. The resulting morphologically changed colonies were screened for alkaloid production and quali- tative changes of alkaloid spectrum. Culture media: Seed-stage medium was modified after ARCAMOMet al. (1961) and composed of 40 g/l mannitol, 10 g/l glucose, 10 g/l succinic acid, 3 g/l KH,PO,, 0,3 g/1 MgSO, . 7 H,O and 2 g/1 chick pea-meal in tap water; the pH of 5.2 was adjusted with ammonia before sterilization. Production-stage media: Three different media were used for the production cultures: Medium P-2: 150 g/l mannitol and 50 g/l peptone “Torlak” in distilled water; pH was about 6.8 without regulation (SOX er al. 1982). Medium MCK was modified after PERTOTet al. (1984) and composed of 50 g/l mannitol, 10 g/1 citric acid, 5 g/1 yeast extract “Torlak”, 1 g/1 KH,PO,, 0.3 g/1 MgSO, . 7 H,O and 20 g/l NaCl in tap water; the pH of 5.2 was adjusted with ammonia. Medium D (KOBELet al. 1964): 50 g/1 sorbitol. 36 g/1 succinic acid, 2 g/1 KH,PO,, 0.3 g/l MgSO, . 7 H,O, 1 mg FeSO, . 7 H,O and 10 mg/l ZnSO, 7 H,O in distilled water and was adjusted to a pH of 5.4 with NH,OH. All culture media were sterilized for 25 min at 121 “C, except medium D which was sterilized for 20 minutes at 108 “C. Culture conditions: C.paspali strains, both the parent and the mutant strain, were maintained on potato- glucose agar at 24 ’C and 14-21-day colonies were used for inoculating the seed medium. Screening experiments were carried out in two stages : seed-stage fermentation and production-stage fer- mentation. The 6-day old cultures were homogenized in a WARINGblendor for 10 sec and 10-15 % (V/V) were used to inoculate the production medium. The production-stage cultures were harvested after 15 days. The alkaloid content as well as the composition of the alkaloid complex were then determined. Submerged cultures were incubated on a rotary shaker (frequency 4 Hz, 50 mm stroke, 24 “C). Analytical methods: Mycelial dry weight: Culture broth (50 ml) was filtered, washed twice with water and dried at 85 “C to constant weight. Total alkaloids: Culture filtrate suitably diluted (2 ml) was mixed with VAN URKreagent (4 ml), which was prepared as described by AGURELL(1966), and the blue color determined spectrophotometrically with refe- rence to a standard solution of ergometrine (free base). The alkaloid composition was evaluated by thin-layer chromatography according to GROGERand ERGE (1963) and scanned on a CAMAGTLC scanner 11. Results and discussion The Claviceps paspali CP-2 mutant isolated after UV irradiation significantly differs from the original strain. Differences can be observed in morphological characteristics of colonies and differentiation of submerged mycelium as well as in the composition of the alkaloid spectrum. Colonies of the mutant, grown on potato-glucose agar have a smooth surface and white fluffy aerial mycelium, covered with yellow or greenish droplets of the exudate (“honeydew”), while colonies of the parent strain are compact with an even border, without fluffy aerial hyphae and without honeydew droplets (Fig. I). These morphological differences facilitated the selection. No conidia or arthrospores could be observed in the mutant’s honeydew droplets as it was noticed in some other C. puspali strains (RYLKOet a/. 1988). We have found that honeydew droplets contain polysaccharides as also reported for other Claviceps strains (RICKOVAet a/. 1986). Further we have found that polysaccharides are composed mainly of arabinose, galactose and xylose. The composition of sugars in the honeydew depends very probably on the composition of the medium used. In submerged cultures the mycelium of the mutant differentiated more or less into swollen, arthrosporoid-like cells and showed a tendency to fragmentize (Fig. 2). Such a morphological differentiation has never been observed with the wild strain of C. paspuli which retains filamentous structure throughout the fermentation. An alkaloid-blocked mutant of Cluviceps puspuli 53 Fig. 1 Colonies of C. paspali: parent strain (left) and mutant strain (right) Fig. 2 Differentiated hyphae of a C. paspali mutant strain in submerged culture 54 ELIZABETAPERTOT et al. Table 1 Percent content of individual alkaloids comprising the total alkaloid mixture of the parent and mutant strain Alkaloid composition Media Parent strain Blocked-mutant P-2 D MCK P-2 D MCK Lysergole 1.8 0.59 0.71 2.16 2.21 6.65 Lysergic acid amide 14.75 10.64 23.72 31.56 9.47 36.65 a-hydroxyethyl amide of lysergic acid 28.55 22.5 29.87 41.55 18.60 44.97 Ergometrine 32.5 I 2.82 24.59 - - - Ergometrinine 6.06 2.7 11.43 - - - Isolysergic acid amide 8.31 33.7 4.5 14.08 38.06 6.36 a-hydroxyethyl amide ' of isolysergic acid 5.22 26.29 3.45 10.32 27.71 5.09 Total alkaloids mg/I 2646 1028 1644 1552 1147 1257 Table 2 Biosynthetic activity of the parent strain and isolated mutant in submerged cultures in different media Medium P-2 Medium D Medium MCK Biomass Alkaloids Biomass Alkaloids Biomass Alkaloids g/l mgb s/l mg/l g/l mg/l Parent strain 20.17 2647 9.63 1028 12.97 1644 Mutant strain 21.52 1552 14.43 1147 15.30 1257 Differences in the alkaloid composition between the CP-2 mutant and the parent strain are also significant and are presented in Table 1 where each value represents the arithmetic mean value of at least 12 individual cultures. The alkaloid composition varies somehow with the medium composition ; as can be seen from Table 1, in the P-2 and MCK media the 1-series simple derivatives of lysergic acid prevail while the medium D favours formation of d-forms, isolysergic acid derivatives, which are less interesting. As known, many compounds in the lysergic acid series exlubit pronounced pharmacological effects but the corresponding isolysergic acid derivatives are almost completely devoid of pharmacological activity. The main alkaloid components produced.'by the parent Claviceps paspali strain are the 1- and d-forms of ergometrine, lysergic acid a-hydroxyethylamide and lysergic acid amide. The CP-2 mutant synthesizes neither ergometrine nor ergometrinine in all three different nutrient media used, though the parent strain produced a significant quantity of both, particularly in the P-2 and MCK media. These results clearly indicate a block in the mutant's biosynthetic pathway. The amide components of ergometrine and lysergic acid cr-hydroxyethylamide are derived from L-alanine (GR~GERet al. 1968), the presumable intermediate being lysergylal- anine (REHACEK,1983).
Load more

Recommended publications
  • Risk Assessment of Argyreia Nervosa
    Risk assessment of Argyreia nervosa RIVM letter report 2019-0210 W. Chen | L. de Wit-Bos Risk assessment of Argyreia nervosa RIVM letter report 2019-0210 W. Chen | L. de Wit-Bos RIVM letter report 2019-0210 Colophon © RIVM 2020 Parts of this publication may be reproduced, provided acknowledgement is given to the: National Institute for Public Health and the Environment, and the title and year of publication are cited. DOI 10.21945/RIVM-2019-0210 W. Chen (author), RIVM L. de Wit-Bos (author), RIVM Contact: Lianne de Wit Department of Food Safety (VVH) [email protected] This investigation was performed by order of NVWA, within the framework of 9.4.46 Published by: National Institute for Public Health and the Environment, RIVM P.O. Box1 | 3720 BA Bilthoven The Netherlands www.rivm.nl/en Page 2 of 42 RIVM letter report 2019-0210 Synopsis Risk assessment of Argyreia nervosa In the Netherlands, seeds from the plant Hawaiian Baby Woodrose (Argyreia nervosa) are being sold as a so-called ‘legal high’ in smart shops and by internet retailers. The use of these seeds is unsafe. They can cause hallucinogenic effects, nausea, vomiting, elevated heart rate, elevated blood pressure, (severe) fatigue and lethargy. These health effects can occur even when the seeds are consumed at the recommended dose. This is the conclusion of a risk assessment performed by RIVM. Hawaiian Baby Woodrose seeds are sold as raw seeds or in capsules. The raw seeds can be eaten as such, or after being crushed and dissolved in liquid (generally hot water).
    [Show full text]
  • Phoretic Analysis of Ergot Alkaloids. Relations Mobility in the Cle Vine
    Acta Pharm, Suecica 2, 357 (1965) Thin-layer chromatographic and thin-layer electro- phoretic analysis of ergot alkaloids.Relations between structure, RM value and electrophoretic mobility in the cle vine series STIG AGUREll DepartMent of PharmacOgnosy, Kunql, Farmaceuliska Insiitutei, StockhOLM, Sweden SUMMARY A thin-layer chromatographic and electrophoretic study of the ergot alkaloids has been made, to find rapid methods for the separation and identification of the known ergot alkaloids. The mobilities of ergot alkaloids in several useful chromatographic and electrophore- tic systems are recorded. Relations have been observed between structure and R" value in methanol-chloroform on Silica Gel G. A simple, rapid thin-layer electrophoretic technique has been de- vised for separation of ergot alkaloids, and a relation between structure and electrophoretic mobility is evident. Two-dimensional combinations of thin-layer chromatography and thin-layer electro- phoresis and chromatography are described. Numerous paper chromatographic procedures have been published for separation of the ergot alkaloids and their derivatives. Hofmann (1) and Genest & Farmilio (2) have recently listed these systems. The general advantages of thin-layer chromatography (TLC) over paper partition chromatography are well known: shorter time of equilibration and devel- opment, generally better resolution, smaller amounts of substance rc- quired, and wider choice of reagents. Several reports of TLC of ergot alkaloids have been published. In gene- ral, these investigations (2-6 and others) have dealt 'with limited groups of alkaloids, or with a specific problem involving at most a dozen of the 40 now known naturally occurring ergot alkaloids. Some paper chromate- .357 graphic systems using Iorrnamide-treated papers have also been adopted for thin-layer chromatographic use (7, 8).
    [Show full text]
  • Electronic Supplementary Material (ESI) for RSC Advances. This Journal Is © the Royal Society of Chemistry 2017
    Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2017 The physicochemical properties and NMR spectra of some ergot alkaloids are summarized as following. Especially, under the influence of acids, alkaloids or light, the carbo at the position 8 of D-lysergic acid derivatives can occur isomerization which 8R is changed into 8S to form the responding isomers, lead to the formation of α-ergotaminine, ergocryptinine, ergocorninine and ergocristinine which the NMR data are shown in Table S1, S2 and S3. Clavine Agroclavine: crystal (acetone), mp 198~203℃, soluble in benzene, ethanol, slightly soluble 1 in water. Molecular formula: C16H18N2, MW m/z: 238. The data of H-NMR (Pyridine-d5，220 13 MHz) and C-NMR (Pyridine-d5，15.08 MHz) {Bach, 1974 #48}are shown in Table S1 and S2. Elymoclavine: mp 250~252℃, Molecular formula: C16H18N2O, MW m/z: 254. The data of 1 13 H-NMR (CDCl3, 220 MHz) and C-NMR (CDCl3, 15.08 MHz){Bach, 1974 #48} of its acetate derivatives are shown in Table S1 and S2. 1 Festuclavine: mp 241℃, Molecular formula: C16H18N2, MW m/z: 238. The data of H-NMR 13 (CDCl3, 220 MHz) and C-NMR (CDCl3, 15.08 MHz) {Bach, 1974 #48}are shown in Table S1 and S2. Fumigaclavine B: mp 265~267℃, Molecular formula: C16H20N2O, MW m/z: 256. The data of 1 13 H-NMR (pyridine-d5，220 MHz) and C-NMR (pyridine-d5，15.08 MHz) {Bach, 1974 #48}are shown in Table S1 and S2. Ergoamides Ergometrine: white crystal (acetone), mp 162℃.
    [Show full text]
  • Ergometrine Maleate
    The European Agency for the Evaluation of Medicinal Products Veterinary Medicines Evaluation Unit EMEA/MRL/237/97-FINAL June 1999 COMMITTEE FOR VETERINARY MEDICINAL PRODUCTS ERGOMETRINE MALEATE SUMMARY REPORT 1. Ergometrine is a naturally occurring alkaloid found in ergot (Claviceps purpurea). It is classified as a water-soluble lysergic acid derivative, and is an orally-active stimulant of uterine contractions. The maleate salt (ergometrine maleate) exhibits greater stability than the free base and is the usual form in which the alkaloid is used in medicinal products. It is used in veterinary medicine in the control of postpartum uterine haemorrhage, removal of fluid from atonic uteri, to prevent pro-lapsed uteri, and judiciously in terms of timing to aid in suturing the uterus after caesarean section or in replacing an everted uterus. Dose regimens are: cows and mares: 2 to 5 mg/animal (intravenously or intramuscularly); ewes, goats and sows: 0.5 to 1 mg/animal (intramuscularly). In human medicine, it is used orally and parenterally in the prevention and treatment of postpartum haemorrhage caused by uterine atony and for the stimulation of uterine involution. Usual oral doses are 500 µg 3 times daily up to 1.8 mg daily (approximately 0.03 mg/kg bw). Ergot alkaloids have been reported to be present in flour from rye, wheat and barley in amounts ranging from 0.01 to 2.36 mg/kg flour. EU legislation restricts the maximum percentage of ergot tolerated in flour to 0.1%. Total daily human intake of ergot alkaloids from contaminated foodstuffs of plant origin has been estimated as up to 7.8 µg/person.
    [Show full text]
  • Eurofins IAC Ergot Alkaloids 3Ml Product Code: EFLS3275 Tecna Code:IA768
    Instruction of the Clean-up Process Using Eurofins IAC Ergot Alkaloids 3ml Product Code: EFLS3275 Tecna code:IA768 THE PRESENT DOCUMENT IS A FAC-SIMILE VERSION. When performing the assay, please use the kit insert. Clean-up of Commodity Extracts of Food and Feed Samples containing 12 Priority Ergot Alkaloids Via Immunoaffinity Chromatography and Subsequent HPLC Analysis with Fluorescence Detection 1. Target of 12 priority Ergot Alkaloids: The most abundant ergot alkaloides produced by fungus Claviceps purpurea comprise six pairs of epimeric ergot alkaloides which can be classified as priority ergot alkaloides1. They form the target of Eurofins IAC Ergot Alkaloids 3ml column. C8-(R)-Epimers (toxic) C8-(S)-Epimers (not toxic) Ergometrine Ergometrinine Ergotamine Ergotaminine Ergosine Ergosinine Ergocornine Ergocorninine Ergocryptine Ergocryptinine Ergocristine Ergocristinine Because both epimers can change to each other under certain conditions, e.g. under acidic or basic influence, in nature and/or during sampling/extraction/analysis, the non-toxic C8-(S)-epimers have to be included in determination as well. 2. Principle: This instruction of priority ergot alkaloid determination in food and feed focuses on the enrichment step of extract using immunoaffinity column (IAC) and quantification with HPLC. Accepted laboratory extraction and IAC treatment methods could essentially be maintained. Full performance of the IAC column is given if pronounced criteria of organic solvent tolerance, elution process and working range of Eurofins IAC Ergot Alkaloids 3ml column is followed. Many pretreatment methods of ergot alkaloids determination in food and feed show low sensitivity because of interfering substances if problematic matrices are applied. This method of content determination of ergot alkaloids combines the high selectivity of an immunoaffinity column (IAC) with its potential to concentrate analytes and additional step of purification by HPLC column.
    [Show full text]
  • A COMPARISON Oit SOME UI TEE Being
    A COMPARISON OIt SOME UI TEE ERGOT ALKALOIDS. being a Thesis Presented for the Degree of Doctor of Medicine Edinburgh University by Adam Cairns White, ßß.B., Ch.B., Ph.D., Wellcome Physiological Research Laboratories, Langley Court, Beckenham, Kent. INS, September, 1939. Com-parison alLome FrEot Alkaloids. A- Section I. Contents. Introduction pp 1-8 General Toxic Symptoms in:- Monkey pp. 8 - 27 Fowl pp. 27 - 34 Canary pp. 34 - 36 Ct pp. 36a - 43 Rabbit pp. 43 - 44 Guinea Pig pp. 44 - 45 Mice pp. 45 - 55 Frog p) 55 Discussion of Toxic Symptoms pp. 55 - 60 Actions on Temperature Regulation in:- Mice pp. 60 - 76 Rabbit pp. 77 - 96 -1- The ergot alkaloids, other than those that are molecular compounds, fail into two groups, a laevorotatory physiologically active group, and a dextrorotatory group which is considerably less active. Smith and Timmis drew attention to this fact in their paper on isoergine and isolysergic acids (1). In Table l I have assembled the ergot alkaloids in descending order of the number of constituent carbon atoms, and given, where possible, their rotations in chloroform. The figures are obtained from the papers of Smith and Timmis (1) (2). The decomposition products, ergine and isoergine have also been included. -2- 200 Table 1. (« } 5461 Ergotoxine C35114105N5 -226e Ergotinine 035H4106N5 +446° 191 Ergotinine C35H4106N5 +513° Ergotamine C33H3505N5 -1810 Ergotaminine C33113505N5 +4500 Ergosine C30113705N5 -1930 Ergosinine C30H3705N5 +5220 Ergotnetrine C19x2302N3 - Ergometrinine C19i-12302N3 . +5200 isoErgine C16H170 N3 Ergine C10170 N3 +598° -3- Since the publications of Smith and Timis (1) (2), Stoll and Burckhardt (3) have reported the isolation of yet another large moleculed pair of alkaloids, ergocristine and ergocristinine C35H3905N5 (this is the same gross formula as they give for ergotoxine) for which they have rotations (el )p in chloroform of -186° and +365e respectively.
    [Show full text]
  • Clavine and Lysergic Acid Alkaloids in Varieties of Morning Glory .Pdf
    Phytochcmlatry,1963. Vol. 2, pp. 65 to 70. PtrpamonPress Ltd. Printed in England CLAVINE AND LYSERGIC ACID ALKALOIDS IN VARIETIES OF MORNING GLORY* W. A. TABERand L. C. VINING Prairie Regional Laboratory. National Research Council of Canada. Saskatoon. Saskatchewan and R. A. HEAC~CR Psychiatric Research Unit, University Hospital, Saskatoon, Saskatchewan (Received 16 Judy 1962) Abstract-The seeds of a number of commercially available varieties of Morning Glory (fpomoeu and Convolvulus sp.) habe been found to contain clavine and lysergic acid alkaloids. Using thin layer and paper chromatographic methods, ergine. isoergine, ergometrine, ergometrinine, elymoclavine. penniclavine, and chanoclavine have been tentatively identified. Not all varieties of seed tested contained these alkaloids: one which contained a substantial amount also contained alkaloid in the leaves and stem of the mature plant. INTRODUCTION “OLOLIUQUI” and “Badoh Negro”, the seeds of the convolvulaceous Rivea corymbosa (L.) Hall. f. and Zpomoea violacea (L.), respectively, have long been used by the Aztec Indians to attain a state of mind suitable for divination during traditional religious ceremonies.‘.2 The active principle has only recently been isolated by Hofmann and co-workers; lysergic acid amide (isoergine), a known psychotomimetic, is present and is accompanied in the seed by ergine, elymoclavine, charm&vine and lysergol as well as other related substances in smaller amounts.8*4ss Subsequent studies in our laboratorie&’ showed that the alkaloids are present in the microbially sterile embryo, and also in the leaf and stem, but not the root, of the mature plant. From this evidence it was considered reasonably certain that the alkaloids are a true metabolic product of the plant and not of an invading microbial parasite or contaminant.
    [Show full text]
  • 7. Ergot Alkaloids and Other Metabolites of the Genus Claviceps
    7. ERGOT ALKALOIDS AND OTHER METABOLITES OF THE GENUS CLAVICEPS MARTIN BUCHTA and LADISLAV CVAK Galena a.s. Opava, 747 70 Czech Republic 7.1. INTRODUCTION Both parasitic and saprophytic ergot is a producer of vast number of compounds from which alkaloids represent the most important group. Over 80 ergot alkaloids (EA) have as yet been isolated from diverse natural material, mainly from Claviceps strains, but also from other fungi and higher plants. Besides alkaloids and some other secondary metabolites (pigments and mycotoxins), ergot produces also some unusual primary metabolites of the lipidic and sacharidic nature. 7.2. ERGOT ALKALOIDS Only alkaloids isolated from different Claviceps species are described in this chapter. EA produced by other fungi and higher plants are the subject of the Chapter 18. Beside this limitation, nearly 70 ergot alkaloids, produced by genus Claviceps, are discussed here. 7.2.1. Ergot Alkaloid Structure All the EA are biosynthetically derived from L-tryptophane, that means indole is the base of their structure. Although the most of EA contains a tetra-cyclic ergoline system (Figure 1), there are some exceptions whose structure is created only by two or three cycles. Natural alkaloids can be divided into three groups according to their structure: The first, structurally simplest group, contains alkaloids of clavine type, the second group are simple amides of lysergic and paspalic acids and the third, most complex group is created by the alkaloids of peptidic type. Biosynthetically, the peptidic EA can be understand as tetrapeptides containing lysergic acid as the first member of the peptidic chain. The other amino acids of the tetrapeptide are variable, which provides the basis for a great diversity of this group of EA.
    [Show full text]
  • Cleaving Ergot Alkaloids by Hydrazinolysis—A Promising Approach for a Sum Parameter Screening Method
    toxins Article Cleaving Ergot Alkaloids by Hydrazinolysis—A Promising Approach for a Sum Parameter Screening Method Maximilian Kuner 1, Susanne Kühn 2, Hajo Haase 3 , Klas Meyer 1 and Matthias Koch 1,* 1 Bundesanstalt für Materialforschung und-prüfung (BAM), 12205 Berlin, Germany; [email protected] (M.K.); [email protected] (K.M.) 2 Institut Kirchhoff Berlin GmbH, 13347 Berlin, Germany; [email protected] 3 Department of Food Chemistry and Toxicology, Technische Universität Berlin, 10623 Berlin, Germany; [email protected] * Correspondence: [email protected] Abstract: Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their cor- responding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods—acidic esterification and hydrazinolysis—are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40-60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix Citation: Kuner, M.; Kühn, S.; Haase, solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected.
    [Show full text]
  • List of Investigated Metabolites and Respective Limits of Detection
    Appendix: List of investigated metabolites and respective limits of detection Analyte LOD (µg/kg sample) 15-Acetyldeoxynivalenol 53 15-Hydroxyculmorin 18 15-Hydroxyculmoron 110 15-Methyl_epi_Fumiquinazolin A N/A 15-Monoacetoxyscirpenol 7 16-Ketoaspergillimide 2 2-Chlorunguinol 0,3 3-Acetyldeoxynivalenol 5 3-Nitropropionic acid 4 3-O-Methylvirdicatin 1 4-Hydroxyalternariol 9 4-Monoacetoxyscirpenol N/A 5-Hydroxyculmorin 180 5-Methylmellein 26 7-Hydroxypestalotin 2 A 23187 N/A AAL TA Toxin 21 Abscisic acid 7 Acetyldeoxypentahydroxyscirpenol N/A Acetylpentahydroxyscirpenol N/A Aflatoxicol 9 Aflatoxin B1 1 Aflatoxin B2 2 Aflatoxin G1 1 Aflatoxin G2 4 Aflatoxin M1 2 Aflatoxin M2 N/A Aflatoxin P1 11 Aflatrem N/A Aflavarin N/A Agistatin B 35 Agistatin D 25 Agistatin E 12 Agroclavine 1 Aigualomycin D 9 Alamethicin F30 4 alpha-Zearalenol 4 alpha-ZOL-Glucosid 11 Alteichin N/A Altenuene 2 Altenusin 280 Alternarian acid 350 Alternariol 2 Alternariol-3-glucoside 4 Alternariol-9-glucoside 4 Alternariolmethylether 0,1 Alternariolmethylether-glucoside 35 Altersetin 2 Altersolanol 110 Altertoxin-I 4 Altertoxin II N/A Amidepsin B 5 Aminooctadecyldecan-3-ol 21 Amoxycillin N/A Amphotericin N/A Anacin 28 Andrastin A 1 Andrastin B 2 Andrastin C N/A Andrastin D N/A Anisomycin 1 Anomalin A N/A Antibiotic F 1849 A 4 Antibiotic L 696474 71 Antibiotic PF 1052 7 Apicidin 1 Ascochlorin 1 Ascomycin 11 Asparason A N/A Aspercolorin 4 Asperfuran 71 Aspergamide A 9 Aspergillicin Derivat 4 Aspergillin PZ N/A Aspergillimide 1 Asperglauccide 0,4 Asperlactone 0,1 Asperloxin
    [Show full text]
  • Validation of Ergot Alkaloids in Feeds by LC-MS/MS AAFCO Annual Meeting 2018 Mélanie Titley & Lise-Anne Prescott Ottawa Laboratory (Carling)
    Validation of Ergot Alkaloids in Feeds by LC-MS/MS AAFCO Annual Meeting 2018 Mélanie Titley & Lise-Anne Prescott Ottawa Laboratory (Carling) © 2017 Her Majesty the Queen in Right of Canada (Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited. Overview • CFIA - OLC - FFC • What are Ergot Alkaloids (EA)? • Upcoming Regulations • Method Summary • Validation Results • Challenge - Standards • Next Steps CFIA - OLC - FFC • Regulatory analytical testing for CFIA enforcement and monitoring programs • Accredited for method development, validation and evaluation to meet CFIA’s changing needs • Expert scientific advice to Program officials, industry, other laboratories, national and international regulatory and scientific organizations What are Ergot Alkaloids? • Ergot Infection • Rye, triticale, wheat, barley, oats and grasses • Ergotism • What causes ergotism • Effect on humans • Effect on animals Dominique Jacquin, https://en.wikipedia.org/wiki/Ergotis m What are EAs? • The most common ergot alkaloids produced by Claviceps purpurea • Ergocornine, Ergocorninine • Ergocristine, Ergocristinine • Ergocryptine, Ergocryptinine • Ergometrine, Ergometrinine • Ergosine, Ergosinine • Ergotamine, Ergotamine Ergometrine (www.chemspider.com) • Epimers r-isomers (-ine) are biologically active • Epimers s-isomers (-inine) are less active Upcoming Regulations Proposed and current maximum levels for total ergot alkaloids Proposed Maximum Limit: Proposed Maximum Current Action Single Ingredient Limit: Level: Feeds (e.g.,
    [Show full text]
  • Effects of Continuously Feeding Diets Containing Cereal Ergot Alkaloids on Nutrient Digestibility, Alkaloid Recovery in Feces, and Performance Traits of Ram Lambs
    toxins Article Effects of Continuously Feeding Diets Containing Cereal Ergot Alkaloids on Nutrient Digestibility, Alkaloid Recovery in Feces, and Performance Traits of Ram Lambs Stephanie Coufal-Majewski 1, Kim Stanford 2,* ID , Tim McAllister 3, Yuxi Wang 3, Barry Blakley 4, John McKinnon 5, Mary Lou Swift 6 and Alexandre V. Chaves 1 ID 1 Faculty of Science, School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia; [email protected] (S.C.-M.); [email protected] (A.V.C.) 2 Livestock Research Section, Alberta Agriculture and Forestry, Lethbridge, AB T1J 4V6, Canada 3 Science and Technology Branch, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1, Canada; [email protected] (T.M.); [email protected] (Y.W.) 4 Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada; [email protected] 5 Department of Animal Science, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada; [email protected] 6 Ruminant Nutrition, Hi-Pro Feeds, Okotoks, AB T1S 1A2, Canada; [email protected] * Correspondence: [email protected]; Tel.: +1-403-381-5150 Received: 26 October 2017; Accepted: 14 December 2017; Published: 19 December 2017 Abstract: Allowable limits for cereal ergot alkaloids in livestock feeds are being re-examined, and the objective of this study was to compare nutrient digestibility, growth performance and carcass characteristics of ram lambs fed a range of alkaloid concentrations, including the maximum currently allowed in Canada (2 to 3 ppm). Four pelleted diets were fed: control, with no added alkaloids; 930; 1402; and 2447 ppb alkaloids based on total R and S epimers.
    [Show full text]